Mono-oxygenases catalyse the selective oxidation of non-functionalised hydrocarbons using oxygen.sup.1, and are therefore of great interest for potential use in organic synthesis. However, progress in this area has been hampered by the difficulty in isolating sufficient quantities of enzyme and the associated electron-transfer proteins. Despite the availability of amino acid sequences of more than 150 different cytochrome P-450 mono-oxygenases, to date structural data of only three are available.sup.2,3,4, and few have been successfully over-expressed in bacterial systems.sup.5.
One cytochrome P-450 mono-oxygenase, which is soluble and can be expressed in sufficient quantities, is the highly specific P-450.sub.cam from P.putida which catalyses the regio- and stereo-selective hydroxylation of camphor (1) to 5-exo-hydroxycamphor.sup.6. The high resolution crystal structure of P-450.sub.cam has been determined.sup.2, and since the mechanism of action of this bacterial enzyme is believed to be very similar to that of its mammalian counterparts, it has been used as a framework on which models of mammalian enzymes are based.
The nucleotide sequence and corresponding amino acid sequence of P-450.sub.cam have been described.sup.5. The location of an active site of the enzyme is known and structure-function relationships have been investigated.sup.13, 14. Mutants of P-450.sub.cam have been described, at the 101 and 185 and 247 positions.sup.15, and at the 87 position.sup.16. A mutant in which tyrosine 96 has been changed to phenyl alanine-96 has been described.sup.12,17,18. But in all these cases the papers report effects of the mutations on the mechanisms of known oxidation reactions. There is no teaching or suggestion that mutation might be used to provide biocatalysts for oxidation of different substrates.